E3 ubiquitin ligase RNF180 prevents excessive PCDH10 methylation to suppress the proliferation and metastasis of gastric cancer cells by promoting ubiquitination of DNMT1

Background Downregulation of certain tumor-suppressor genes (TSGs) by aberrant methylation of CpG islands in the promoter region contributes a great deal to the oncogenesis and progression of several cancers, including gastric cancer (GC). Protocadherin 10 (PCDH10) is a newly identified TSG in various cancers and is downregulated in GC; however, the specific mechanisms of PCDH10 in GC remain elusive. Here, we elucidated a novel epigenetic regulatory signaling pathway involving the E3 ubiquitin ligase RNF180 and DNA methyltransferase 1 (DNMT1), responsible for modulating PCDH10 expression by affecting its promoter methylation. Results We revealed that PCDH10 was downregulated in GC cells and tissues, and low PCDH10 expression was correlated with lymph node metastasis and poor prognosis in patients with GC. Additionally, PCDH10 overexpression suppressed GC cell proliferation and metastasis. Mechanistically, DNMT1-mediated promoter hypermethylation resulted in decreased expression of PCDH10 in GC tissues and cells. Further analysis revealed that RNF180 can bind directly to DNMT1 and was involved in DNMT1 degradation via ubiquitination. Additionally, a positive correlation was found between RNF180 and PCDH10 expression and an inverse association between DNMT1 and PCDH10 expression showed considerable prognostic significance. Conclusion Our data showed that RNF180 overexpression upregulated PCDH10 expression via ubiquitin-dependent degradation of DNMT1, thus suppressing GC cell proliferation, indicating that the RNF180/DNMT1/PCDH10 axis could be a potential therapeutic target for GC treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s13148-023-01492-y.

University. Another GC cell lines AGS and N87 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All GC and GES-1 cell lines, excluding AGS cell lines were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (OriCell, New Zealand) and 1% penicillinstreptomycin (Gibco, USA). AGS was cultured in F12 Nutrient Mixture medium (Gibco, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All cells were cultured at 37 °C in a thermostatic incubator containing 5% CO2. All cell lines were authenticated by STR profiling within three years, and the experiments were conducted using mycoplasma-free cells.

qRT-PCR
According to the manufacturer's instructions, total RNA was extracted from tissues and cultured cells with TRIzol reagent (Invitrogen, USA) and reverse-transcribed into cDNA using a PrimeScript RT Reagent Kit (Takara, Japan). The cycling conditions were 94 °C for 5 min, 40 cycles of 95•C for 5 s, 60•C for 20 s, and 95•C for 15 s, 60•C for 1min, 95•C for 30 s, 60•C for 15 s. Triplicates were performed for each reaction. The primer sequences for qRT-PCR are shown in Additional file 1: Table S4.

Western blotting
Whole-cell extracts were prepared by lysing cells in RIPA buffer (Millipore, USA) supplemented with phosphatase inhibitor and protease inhibitor (Millipore, USA). Western blots and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to separate protein lysates and detect target proteins. The primary antibodies used are listed in Additional file 1: Table S5.

Gene chip detection analysis
Stable HGC27-RNF180 cells and HGC27-vector cells were were lysed in Trizol reagent (Invitrogen) and total RNA was extracted according to instructions. The global mRNA expression profiles were detected using a GeneChip® PrimeView™ Human Gene Expression Array (Affymetrix, Santa Clara, CA, USA). The raw data were analyzed using GeneSpring software version 12.5 (Agilent Technologies, Santa Clara, CA, USA) and normalized using a quantile algorithm. Gene expression changes ≥ 2.0-fold higher or lower and P < 0.05 were considered as biologically significant. Differences in mRNA expression were verified by qRT-PCR.

RNA sequencing (RNA-seq) and analysis
Three replicates for stable HGC27-PCDH10 and HGC27-vector cells were seeded and collected and total RNA was extracted using the RNeasy kit (Qiagen, Germany) according to the manufacturer's instructions. RNA integrity was examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Following the sample integrity check, the subsequent analysis was conducted only for samples with integrity ≥ 7. Library construction and transcriptome sequencing and analysis were performed by OE Biotech Co., Ltd. (Shanghai, China). Thresholds for significantly differential expression were set as |log2FoldChange|<1 and P < 0.05.

Next-generation sequencing (NGS) approach for methylation analysis
Total DNA was extracted using a QIAamp® DNA Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. Raw reads were filtered according to two steps: 1) Removing adaptor sequence if reads contains by cutadapt (v 1.2.1); 2) Removing low quality bases from

Cell Counting Kit-8 (CCK-8) and colony-formation assays
Briefly, for cell proliferation, cells were inoculated into 96-well plates (1000 cells per well) and 10 µl of CCK-8 reagent was added to the wells after 1, 2, 3, and 4 days. The plates were incubated for 2 h, and the absorbance was determined at 490 nm. For colony-formation assays cells were seeded in a 6-cm culture dish (750 cells) and the culture medium was refreshed every 3 days for 2 weeks. The cells were then washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and stained with 0.4% crystal violet for 15 min. The number of colonies containing >10 cells was counted manually and averaged over duplicate wells.

Cell cycle and apoptosis
Stable gastric cancer cells were collected, washed with phosphate-buffered saline twice, and stained with propidium iodide (Sigma-Aldrich) at a final concentration of 17.4 lg/mL. Cells were analyzed using fluorescenceactivated cell sorting analysis (FACS Calibur; BD Biosciences, San Jose, CA), and data were analyzed using ModFit LT software (Verity Software House, Topsham, ME). The apoptosis analysis was determined by PE Annexin V kit (PharMingen, BD Biosciences). Cells were stained with PE Annexin V and 7-AAD, and data were analyzed using EXPO32 ADC Cellquest Analysis Software (Beckman Coulter, Brea, CA).

Cell migration and invasion assay
Cell migration and invasion was assayed using Transwell chambers (6.5 mm; Corning, NY,

Protein half-life detection
HGC27 cells were infected with the lentiviral vector encoding RNF180 (LV-RNF180) or with negative control vector, and MKN45 cells were infected with lentiviral vectors carrying RNF180-shRNA or empty vector, as described above. A total of 10 mg/mL cycloheximide (HY-12320, MCE) was added to the culture medium. Following exposure to cycloheximide, cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors at the indicated time points. Furthermore, cells were also incubated with 10 μM MG132 (HY-13259, MCE) for an additional 10 h and then collected for western blotting to determine the amount of DNMT protein.

Immunohistochemical (IHC) staining
Tissue staining was performed as previously published. 3

Animal experiments
Xenograft experiments in nude mice were approved by the Animal Experimentation Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. GC cells stably expressing PCDH10 and control cells were harvested and suspended in PBS. A total of twelve female BALB/c mice between 4 and 6 weeks were randomly divided into two groups, each with six mice, and subcutaneous injections of 4×106 cells were administered to each mouse in the inguinocrural region. Measurements of tumor dimensions were taken manually three times a week, and tumor volumes calculated using the formula (width2 x length/2).
Approximately 4 weeks after injection, the mice were sacrificed, and the tumors were